The p53 gene is mutated in over 50 different types of human cancers, including familial and spontaneous cancers, and is believed to be the most commonly mutated gene in human cancer (Zambetti and Levine, FASEB (1993) 7:855-865; Hollstein, et al., Nucleic Acids Res. (1994) 22:3551-3555). Greater than 90% of mutations in the p53 gene are missense mutations that alter a single amino acid that inactivates p53 function. Aberrant forms of human p53 are associated with poor prognosis, more aggressive tumors, metastasis, and short survival rates (Mitsudomi et al., Clin Cancer Res 2000 October; 6(10):4055-63; Koshland, Science (1993) 262:1953).
The human p53 protein normally functions as a central integrator of signals including DNA damage, hypoxia, nucleotide deprivation, and oncogene activation (Prives, Cell (1998) 95:5-8). In response to these signals, p53 protein levels are greatly increased with the result that the accumulated p53 activates cell cycle arrest or apoptosis depending on the nature and strength of these signals. Indeed, multiple lines of experimental evidence have pointed to a key role for p53 as a tumor suppressor (Levine, Cell (1997) 88:323-331). For example, homozygous p53 “knockout” mice are developmentally normal but exhibit nearly 100% incidence of neoplasia in the first year of life (Donehower et al., Nature (1992) 356:215-221).
The biochemical mechanisms and pathways through which p53 functions in normal and cancerous cells are not fully understood, but one clearly important aspect of p53 function is its activity as a gene-specific transcriptional activator. Among the genes with known p53-response elements are several with well-characterized roles in either regulation of the cell cycle or apoptosis, including GADD45, p21/Waf1/Cip1, cyclin G, Bax, IGF-BP3, and MDM2 (Levine, Cell (1997) 88:323-331).
Protein kinases (PKs) play a crucial role in regulating cellular processes, including growth factor response, cytoskeletal changes, gene expression, and metabolism. PKs have very similar sequences and can be grouped based on specificity for the acceptor amino acid. Most PKs phosphorylate either serine/threonine or tyrosine. However, some PKs, referred to as mixed-lineage kinases, have features of both serine/threonine and tyrosine PKs. All PKs have Src homology (SH) domains and can also be grouped as receptors or nonreceptors. Receptor PKs have a transmembrane region, an extracellular ligand-binding domain, and an intracellular catalytic domain.
Mitogen activated protein kinase kinase kinase 12 (MAP3K12), is a dual leucine zipper-bearing kinase, and a member of the mixed lineage protein kinase (MLK) (Reddy, U. and Pleasure, D., (1994) Biochem. Biophys. Res. Commun. 202: 613-620). MAP3K12 contains a COOH-terminal and NH2-terminal proline-rich domains suggestive of src homology 3 (SH3) domain binding regions, and can be autophosphorylated on serine and threonine residues (Holzman, L. et al., (1994) J. Biol. Chem. 269: 30808-30817). This kinase activates the SAPK/JNK signaling pathway, and may play a role in neuronal differentiation (Hirai, S., (1996) Oncogene 12: 641-650).
MAP3K13 protein, also called LZK (leucine zipper-bearing kinase) contains double leucine/isoleucine zippers, has no apparent signal sequence or transmembrane region but does contain a kinase catalytic domain, and an acidic domain at its C-terminal end (Sakuma, H. et al., (1997) J. Biol. Chem. 272: 28622-28629). MAP3K13 shares 86.4% amino acid identity with MAP3K12 and like MAP3K12 it is also a member of the mixed-lineage kinase family of proteins which contain similarities to both serine/threonine and tyrosine kinases (Sakuma, H. et al., (1997) J. Biol. Chem. 272: 28622-28629). These kinases activate the phosphorylation event of c-Jun and turn on JNK-1 (Sakuma, H. et al., (1997) J. Biol. Chem. 272: 28622-28629).
MAP3K12 and MAP3K13 are both highly conserved genes that have been found in organisms from yeast to man. MAP3K12 has been implicated in neuronal cell death (Xu, Z. et al. (2001) Mol Cell Biol 21:4713-24).
The ability to manipulate the genomes of model organisms such as Drosophila provides a powerful means to analyze biochemical processes that, due to significant evolutionary conservation, has direct relevance to more complex vertebrate organisms. Due to a high level of gene and pathway conservation, the strong similarity of cellular processes, and the functional conservation of genes between these model organisms and mammals, identification of the involvement of novel genes in particular pathways and their functions in such model organisms can directly contribute to the understanding of the correlative pathways and methods of modulating them in mammals (see, for example, Mechier B M et al., 1985 EMBO J 4:1551-1557; Gateff E. 1982 Adv. Cancer Res. 37: 33-74; Watson K L., et al., 1994 J Cell Sci. 18: 19-33; Miklos G L, and Rubin G M. 1996 Cell 86:521-529; Wassarman D A, et al., 1995 Curr Opin Gen Dev 5: 44-50; and Booth D R. 1999 Cancer Metastasis Rev. 18: 261-284). For example, a genetic screen can be carried out in an invertebrate model organism having underexpression (e.g. knockout) or overexpression of a gene (referred to as a “genetic entry point”) that yields a visible phenotype. Additional genes are mutated in a random or targeted manner. When a gene mutation changes the original phenotype caused by the mutation in the genetic entry point, the gene is identified as a “modifier” involved in the same or overlapping pathway as the genetic entry point. When the genetic entry point is an ortholog of a human gene implicated in a disease pathway, such as p53, modifier genes can be identified that may be attractive candidate targets for novel therapeutics.
All references cited herein, including sequence information in referenced Genbank identifier numbers and website references, are incorporated herein in their entireties.